Fungal infections are increasingly common as a result of AIDS, transplantation, high dose chemotherapy, steroid treatment, antibiotic treatment, and invasive procedures. However, existing antifungal agents are limited to amphotericin B, flucytosine, and azoles, and drug resistant strains are emerging. We propose to elucidate signal transduction cascades regulating fungal growth and virulence as targets for antifungal drug development. We propose studies on both Candida albicans, the most common human fungal pathogen, and Cryptococcus neoformans, the leading cause of fungal meningitis and an important opportunistic fungal pathogen. We have discovered that the immunosuppressive drugs rapamycin, cyclosporin A, and FK506 have potent antifungal activity. In studies supported by this award, we have identified the fungal drug target proteins, including FKBP12, the Tor1 kinase, and two cyclophilin A homologs. Our studies demonstrate that the antifungal effects of rapamycin are mediated via a complex with FKBP12 that inhibits the fungal Tor1 kinase homolog. We have identified nonimmunosuppressive analogs of each of these drugs that retain antifungal activity. By genetic and biochemical approaches, we show that these analogs take advantage of structural differences between host and fungal enzymes, sparing immune function while impairing fungal cell growth. Finally, we have identified examples of potent synergistic drug interactions. For example, the calcineurin inhibitors cyclosporin A and FK506 are potently synergistic with azoles in Candida albicans. Here we propose to establish the cellular functions and targets of the rapamycin target protein Tor1. The TOR1 gene will be disrupted in diploid strains of C. neoformans to test if it is essential. Targets of the Tor kinase will be identified by genetic and two hybrid screens, and by analyzing gene expression with genome arrays. We will also identify the targets of the C. neoformans cyclosporin A target proteins, the Cpa1 and Cpa2 cyclophilins, which are important for cell growth and virulence. We will determine the molecular basis of synergistic drug interactions. First, we will identify a novel target of the FKBP12-FK506 complex that is synergistic with proton pump inhibitors in C. neoformans. Second, we will focus on the roles of FKBP12 and calcineurin in azole action in C. albicans, testing the hypothesis that calcineurin is either essential or becomes essential during cell membrane stress as a result of azole treatment. Finally, we will test rapamycin and nonimmunosuppressive rapamycin analogs, and the synergistic combination of calcineurin inhibitors and azoles, in animal models of cryptococcosis and candidiasis. Our long term goal is to identify unique targets and develop novel antifungal drug therapies.